title: A thioredoxin reductase and/or thioredoxin system-based mechanism for antioxidant effects of ambroxol.
abstract: Long-term treatment with ambroxol (ABX), a bronchial expectorant, was found to prevent acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The underlying mechanism remains unclear. To address this, we have investigated the effect of ABX on critical antioxidant proteins thioredoxin (Trx) and thioredoxin reductase (TrxR) that are decreased in patients with AECOPD. Trx, TrxR and NADP(H) form Trx system, which is involved in regulating numerous oxidative stress-related events. In human bronchial epithelial cells, treatment with ABX from 0 to 200 μM gradually increased mRNA and protein levels of TrxR/Trx. At these ABX concentrations, TrxR activity was elevated progressively, whereas Trx activity exhibited a dose-dependent biphasic response, increasing at 50 and 75 μM, but decreasing at ABX over 150 μM. Pre-treatment with 75 μM ABX enhanced the capacity of the cells to eliminate reactive oxygen species, which was largely prevented by knockdown of cytosolic Trx (hTrx1). In a purified system, ABX shortened the initial lag phase during the reduction of insulin disulfide by Trx system. Pre-treatment of NADPH-reduced TrxR with ABX caused a dose- and time-dependent increase in thiolate/selenolate species, i.e. the catalytically active form of TrxR. Kinetic analysis demonstrated that the reduction of H2O2 by TrxR or Trx system were enhanced by 100 or 200 μM ABX. When hTrx1 was mixed with ABX in a molar ratio of 1:1 to 1:100 (which could occur in human plasma), changes in intrinsic Trp fluorescence occurred, and the response of reduced hTrx1 was especially remarkable. These data reveal an ABX-sensing mechanism of TrxR/Trx. We therefore conclude that the antioxidant actions of ABX at physiological concentrations are, at least partially, mediated by TrxR and/or Trx system.
authors.affiliation: College of Life Sciences, University of Chinese Academy of Sciences, YuQuan Road 19(A), 100049 Beijing, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Respiratory Medicine, Dayi Hospital Affiliated to Shanxi Medical University, Longcheng Street 99, 030032 Taiyuan, China.
authors.forename: Jianying
authors.initials: J
authors.lastname: Xu
authors.affiliation: School of Public Health, Capital Medical University, 100069 Beijing, China. Electronic address: tianlin@ccmu.edu.cn.
authors.forename: Lin
authors.initials: L
authors.lastname: Tian
authors.affiliation: College of Life Sciences, University of Chinese Academy of Sciences, YuQuan Road 19(A), 100049 Beijing, China. Electronic address: liazho@ucas.ac.cn.
authors.forename: Liangwei
authors.initials: L
authors.lastname: Zhong
mesh_terms: D000551:Ambroxol / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018384:Oxidative Stress; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*; D013879:Thioredoxins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C491203:TXN protein, human; D000551:Ambroxol; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D013880:Thioredoxin-Disulfide Reductase
keywords: 2′,7′-dichlorofluorescin; 2′-7′-dichlorodihydrofluorescein diacetate; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 5,5′-dithiobis-(2-nitrobenzoic acid); 5-(iodoacetamido)fluorescein; 5-IAF; ABX; Ambroxol; DCF; DCFH-DA; DTNB; DTT; Gua HCl; MTT; PMSF; ROS; Reactive oxidative species; Thioredoxin; Thioredoxin reductase; Trx; TrxR; dithiothreitol; guanidine hydrochloride; phenylmethanesulfonyl fluoride; radical oxidative species; thioredoxin; thioredoxin reductase; trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanol, ambroxol